When a sample is exposed to light, light of the same wavelength as that of the illuminating light appears in the form of reflected light and scattered light and, in addition thereto, there are cases where light of wavelengths different from that of the illuminating light is emergent (for example, fluorescence and Raman-scattered light). Useful information about the sample can be obtained by measuring the intensity of this fluorescence or Raman-scattered light, and useful information about the sample can also be obtained by measuring the characteristic of polarization of these beams.
For example, when a sample containing a fluorescent probe is exposed to excitation light and when depolarization of fluorescence emitted from the sample is measured, we can know whether there exists the fluorescent probe bound to a target. More specifically, when the sample containing the fluorescent probe is excited with linearly polarized pulsed light, fluorescence is emitted from the fluorescent probe. The polarization state of the fluorescence is almost linearly polarized at the beginning of exposure to the excitation light; but irregular rotation of the fluorescent probe because of the Brownian motion will disturb the molecular axes of excited fluorescent probe molecules, so that the fluorescence will become gradually depolarized with a lapse of time and a unpolarized state will result finally. The rate of this depolarization of the free fluorescent probe is different from that of the fluorescent probe bound to the target and, therefore, we can know whether the fluorescent probe bound to the target is present, by making use of the difference in the rate of depolarization.
Accordingly, if a two-dimensional image of degrees of this fluorescence depolarization can be measured under a microscope, a location of the fluorescent probe bound to the target can be specified and the behavior or the like of the target in the sample can be analyzed; it is, therefore, expected that this method can contribute to elucidation of various functions in cells, for example. Similarly, it is also expected that useful information concerning the sample can be obtained by measurement of a two-dimensional image of polarization characteristic of the Raman-scattered light under the microscope.
Incidentally, the technique for observing the two-dimensional image of the polarization state of fluorescence is described in D. Axelrod, "Carbocyanine Dye Orientation in Red Cell Membrane studied by Microscopic Fluorescence Polarization," Biophys. J., Vol. 26, pp. 557-574 (1979) and K. Suzuki et al., "Spatiotemporal Relationships Among Early Events of Fertilization in Sea Urchin Eggs Revealed by Multiview Microscopy," Biophys. J., Vol. 68, pp. 739-748 (1995).
The technique described in the paper of D. Axelrod concerns the method for measuring states of polarization of fluorescence generated under irradiation of steady-state light (linearly polarized excitation light), under the microscope and thereby analyzing orientation and mobility of fluorescent dye in a biomembrane. A photodetector used herein is a photomultiplier tube, which obtains a two-dimensional image of fluorescence polarization by scanning the diaphragm on the fluorescent image plane. A problem arising in measuring such fluorescence polarization is measurement errors caused by different responses of the photodetector and the optical system except for the polarizing device to different directions of polarization. In the technique described in this paper, the measurement errors are corrected (polarization response correction) by allowing fairly unpolarized light to pass through a light detecting optical system (an optical system from the sample to the photodetector) and the photodetector.
The technique described in the paper of K. Suzuki concerns the method for splitting the fluorescence emitted from the sample into mutually orthogonal components of linearly polarized light by a polarizing beam splitter, picking up each of images of the two split fluorescent beams by a single camera, and thereby analyzing stationary molecular orientation of the sample. In the technique described in this paper, the polarization response correction is effected by making use of the fact that when a homogeneous sample is excited by unpolarized excitation light, the fluorescence emitted from the sample is of no polarization at all.
However, the techniques described in the paper of D. Axelrod and in the paper of K. Suzuki employ the polarization response correction using the unpolarized light, and the accuracy of correction is low, because this unpolarized light is different from the fluorescence to be observed originally; the measurement accuracy of polarization characteristic is also low accordingly. It is not easy to make ideally unpolarized light.
The present invention has been accomplished in order to solve the above problem and an object of the invention is to provide a polarization characteristic measuring method and apparatus that can measure a characteristic of polarization of a second beam (fluorescence or Raman-scattered light) emitted from a sample irradiated by a first beam (excitation light or illuminating light), with accuracy.